Maternal pregestational diabetes significantly increases risk for congenital malformations, a diabetic complication known as ?diabetic embryopathy?. Animal studies indicate that maternal hyperglycemia-induced oxidative stress is responsible, but specific pathways leading to oxidative stress are incompletely understood. We have used a mouse model of diabetic pregnancy in which malformations are significantly increased in embryos of diabetic mice due to oxidative stress. Oxidative stress results from high rates of glucose transport into embryo cells during maternal hyperglycemia, because embryos express the high KM glucose transporter, GLUT2, during the embryopathy-susceptible period, and GLUT2 expression is necessary for maternal hyperglycemia-induced malformations. We have derived embryonic stem cell (ESC) lines from mouse blastocysts under physiological glucose (5.5mM) and oxygen (5%) conditions that we named LG-ESC (Low Glucose-derived ESC) that retain expression of functional GLUT2 transporters that are not expressed by conventional mouse or human ESC. High glucose media induces oxidative stress in LG-ESC, indicating that these cells can be used to study pathways by which high glucose causes oxidative stress in embryo cells. We hypothesize that regulators of high glucose-induced oxidative stress, when genetically inactivated, will prevent high glucose-induced oxidative stress and can be used to identify pathways that are essential for diabetic embryopathy. In this application, we propose to perform a forward CRISPR-Cas9 screen, using a library containing single guide RNA (sgRNA) sequences targeting the entire mouse genome, to identify genes that are required for high glucose-induced oxidative stress (referred to as, ?HGROSR? (High Glucose Reactive Oxygen Species Regulator) genes) in LG-ESC. Aim 1. Transduce LG-ESC with a lentiviral CRISPR-Cas9 library containing sgRNA targeting the entire mouse genome to identify genes that encode HGROSRs. Following drug selection of transduced cells, they will be cultured in high (25 mM) glucose media to induce oxidative stress. Cells will be incubated with a dye that fluoresces upon conjugating with reduced glutathione (GSH), and then, cells with high fluorescence (low oxidative stress) will be collected by cell sorting. sgRNA sequences that inactivate HGROSR genes in these cells will be identified by sequencing, and data will be analyzed to determine candidate HGROSR genes with multiple enriched sgRNAs. Aim 2. Validate that candidate genes identified in Aim 1 encode HGROSRs. We will transduce LG-ESC with individual sgRNA in lentiviral vectors targeting top candidate genes identified in Aim 1 and (a) confirm inhibition of high glucose-induced oxidative stress, (b) determine the nature (insertion/deletion) of gene inactivation. (c) We will further validate involvement of candidate HGROSR genes in diabetic embryopathy by confirming that they are expressed in mouse embryos during the diabetic embryopathy-susceptible period.